G-CSF is a hormone-like glycoprotein which regulates hematopoiesis and is required for the clonal growth and maturation of normal hematopoietic precursor cells found in the bone marrow; Welte et al., Proc. Natl. Acad. Sci., Vol. 82, pp. 1526-1530 (1985). More specifically, G-CSF, when present in low concentrations, is known to stimulate the production of neutrophil granulocytic colonies when used in vitro G-CSF is also known to enhance neutrophil migration; Gabrilove, J., Seminars in Hematology, Vol. 26, No. 2, pp. 1-4 (1989). Moreover, G-CSF can significantly increase the ability of neutrophils to kill tumor cells in vitro through antibody mediated cellular cytotoxicity; Souza et al., Science, Vol. 232, pp. 61-65 (1986).
In humans, endogenous G-CSF is detectable in blood plasma; Jones et al., Bailliere's Clinical Hematology, Vol. 2, No. 1, pp.83-111. G-CSF is produced by fibroblasts, macrophages, T cells, trophoblasts, endothelial cells and epithelial cells and is the expression product of a single copy gene comprised of four exons and five introns located on chromosome seventeen. Transcription of this locus produces a mRNA species which is differentially processed, resulting in the expression of two forms of G-CSF, one version having a mature length of 177 amino acids, the other having a mature length of 174 amino acids. The form comprised of 174 amino acids has been found to have the greatest specific in vivo biological activity. G-CSF is species cross-reactive, such that when human G-CSF is administered to another mammal such as a mouse, canine or monkey, sustained neutrophil leukocytosis is elicited; Moore et al., Proc. Natl. Acad. Sci., Vol. 84, pp. 7134-7138 (1987).
Human G-CSF can be obtained and purified from a number of sources. Natural human G-CSF (nhG-CSF) can be isolated from the supernatants of cultured human tumor cell lines. The development of recombinant DNA technology, see, for instance, U.S. Pat. No. 4,810,643 (Souza), incorporated herein by reference, has enabled the production of commercial scale quantities of G-CSF in glycosylated form as a product of eukaryotic host cell expression, and of G-CSF in non-glycosylated form as a product of prokaryotic host cell expression.
G-CSF has been found to be useful in the treatment of cancer, as a means of stimulating neutrophil production to compensate for hematopoietic deficits resulting from chemotherapy or radiation therapy. The effective use of G-CSF as a therapeutic agent requires that patients be administered systemic doses of the protein. Currently, parenteral administration via intravenous, intramuscular or subcutaneous injection is the preferred route of administration to humans and has heretofore appeared to be the only practical way to deliver therapeutically significant amounts of G-CSF to the bloodstream, although attempts have been made at oral delivery; see, for example, Takada et al., Chem. Pharm. Bull., Vol. 37, No. 3, pp. 838-839 (1989).
The pulmonary delivery of relatively large molecules is not unknown, although there are only a few examples which have been quantitatively substantiated. Leuprolide acetate is a nonapeptide with luteinizing hormone releasing hormone (LHRH) agonist activity having low oral availability. Studies with animals indicate that inhalation of an aerosol formulation of leuprolide acetate results in meaningful levels in the blood; Adjei et al., Pharmaceutical Research, Vol. 7, No. 6, pp. 565-569 (1990); Adjei et al., InternationaI Journal of Pharmaceutics, Vol. 63, pp. 135-144 (1990).
Endothelin-1 (ET-1), a 21 amino acid vasoconstrictor peptide produced by endothelial cells, has been found to decrease arterial blood pressure when administered by aerosol to guinea pigs; Braquet et al., Journal of Cardiovascular Pharmacology, Vol. 13, suppl. 5, s. 143-146 (1989).
The feasibility of delivering human plasma .alpha.1-antitrypsin to the pulmonary system using aerosol administration, with some of the drug gaining access to the systemic circulation, is reported by Hubbard et al., Annals of Internal Medicine, Vol. III, No. 3, pp. 206-212(1989).
Pulmonary administration of alpha-1-proteinase inhibitor to dogs and sheep has been found to result in passage of some of that substance into the bloodstream; Smith et al., J. Clin. Invest., Vol. 84, pp. 1145-1146 (1989).
Experiments with test animals have shown that recombinant human growth hormone, when delivered by aerosol, is rapidly absorbed from the lung and produces faster growth comparable to that seen with subcutaneous injection; Oswein et al., "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colo., March, 1990. Recombinant versions of the cytokines gamma interferon (IFN-.gamma.) and tumor necrosis factor alpha (TNF-.alpha.) have also been observed in the bloodstream after aerosol administration to the lung; Debs et al., The Journal of Immunology, Vol. 140, pp. 3482-3488 (1988).